The EVDPIGHLY (SEQ ID No: 1) peptide corresponds to amino acid residue numbers 168-176 of the known MAGE-3 protein. The MAGE-3 protein is expressed in many tumour types, including melanomas, and other solid tumours such as Head and Neck Squamous Cell, lung, bladder, gastric and esophageal carcimomas. The MAGE-3 peptide EVDPIGHLY (SEQ ID No: 1) is the best characterised MAGE-3 epitope. It is recognised by both HLA-A1 and HLA-B35 restricted T cells. It is able to elicit cytotoxic activity against peptide-pulsed, HLA-A1 positive target cells, and MAGE-3-expressing HLA-A1 positive melanoma cell lines. The peptide, used as a vaccine, has been shown to induce tumour regression and elicit CTL responses in some of those patients.
Therefore, the EVDPIGHLY (SEQ ID NO: 1) HLA-A1 complex provides a cancer marker that the TCRs of the invention can target. For example, TCRs of the invention may be transformed into T-cells, rendering them capable of destroying tumour cells presenting that HLA complex, for administration to a patient in the treatment process known as adoptive therapy. For this purpose it would be desirable if the TCRs had a higher affinity and/or a slower off-rate for the peptide-HLA complex than native TCRs specific for that complex. Dramatic increases in affinity have been associated with a loss of antigen specificity in TCR gene-modified CD8 T cells, which could result in the nonspecific activation of these TCR-transfected CD8 T cells, so TCRs having somewhat a higher affinity and/or a slower off-rate for the peptide-HLA complex than native TCRs specific for that complex, but not a dramatically higher affinity and/or dramatically slower off-rate for the peptide-HLA than native TCRs, would be preferred for adoptive therapy (see Zhao et al., (2007) J. Immunol. 179: 5845-54; Robbins et al., (2008) J. Immunol. 180: 6116-31; and see also published WO 2008/038002).
TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly, each chain comprises variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region comprises three CDRs (Complementarity Determining Regions) embedded in a framework sequence, one being the hypervariable region named CDR3. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Vα types are referred to in IMGT nomenclature by a unique TRAV number. Thus “TRAV21” defines a TCR Vα region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR. In the same way, “TRBV5-1” defines a TCR Vβ region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence.
The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature.
The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJ regions are often considered together as the joining region.
The α and β chains of αβ TCR's are generally regarded as each having two “domains”, namely variable and constant domains. The variable domain consists of a concatenation of variable region and joining region. In the present specification and claims, the term “TCR alpha variable domain” therefore refers to the concatenation of TRAV and TRAJ regions, and the term TCR alpha constant domain refers to the extracellular TRAC region, or to a C-terminal truncated TRAC sequence. Likewise the term “TCR beta variable domain” refers to the concatenation of TRBV and TRBD/TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBC region, or to a C-terminal truncated TRBC sequence.
The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. The “T cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time-lag, the information therein sometimes needs to be confirmed by reference to the IMGT database.
We have confirmed that a native MAGE-3 TCR (Clone EB81-103 from Dr. Pierre G. Coulie, Cellular Genetics Unit, University of Louvain, Avenue Hippocrate 74, UCL 7459, B-1200 Brussels, Belgium; see also Karanikas, et. al. (2003) “Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus.” J. Immunol. 171(9): 4898-904)) has the following alpha chain and beta chain V, J and C gene usage:
Alpha chain—TRAV21*01/TRAJ28/TRAC (the extracellular sequence of the native MAGE-A3 TCR alpha chain is given in SEQ ID No: 2)
Beta chain:—TRBV5-1*01/TRBD1/TRBJ2-7*01/TRBC2 (the extracellular sequence of the native MAGE-A3 TCR beta chain is given in SEQ ID No: 3). (Note that the TRBV5-1 sequence has 2 allelic variants, designated in IMGT nomenclature as TRBV5-1*01 and *02 respectively, and the native MAGE-A3 TCR clone referred to above has the *01 variation. In the same way, the TRBJ2-7 sequence has two known variations and it is the *01 sequence which is present in the TCR clone referred to above. Note also that the absence of a“*” qualifier means that only one allele is known for the relevant sequence.)
The terms “wild type TCR”, “native TCR”, “wild type MAGE-A3 TCR” and “native MAGE-A3 TCR” are used synonymously herein to refer to this naturally occurring TCR having the extracellular alpha and beta chain SEQ ID Nos: 2 and 3.